Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase

Glutamine synthetase (GS) converts glutamate to glutamine in the presence of ATP and ammonia and requires two divalent metal ions, designated n(1) and n(2), for catalysis. The first intermediate, gamma-glutamyl phosphate, is formed during catalysis by the transfer of the gamma-phosphate of ATP to the gamma-carboxylate of glutamate. Efficient phosphoryl transfer between these two negatively charged moieties is thought to be mediated by the n(2) metal. To explore the role of the n(2) metal in catalysis, histidine 269, a ligand to the n(2) metal, was changed to aspartate, asparagine, glutamate, and glutamine by site-directed mutagenesis. All of the mutants bind two manganese ions as determined by EPR titration. The mutations had little effect on the substrate K-m's except in the case of H269E which exhibited a K-m Glu = 92 mM, a 1000-fold increase compared to that for WT (K-m Glu = 70 mu M). The ability of these mutants to catalyze phosphoryl transfer to glutamate or to the inhibitor phosphinothricin was examined by rapid quench kinetic experiments. Phosphorylated phosphinothricin was detected by P-31 NMR and shown to be produced by both mutants and WT. The rate of phosphoryl transfer to PPT for H269E is reduced 100-fold (0.030 s(-1)) compared to WT (8 s(-1)). The extra negative charge around the n(2) metal ion contributed by glutamate 269 severely reduces the ability of the n(2) metal to mediate efficient glutamate binding in the presence of negatively charged ATP and weakens the interactions between metal ion and the reactants in the transition state, thus resulting in a lower rate of phosphoryl transfer.